ABSTRACT
The time courses of activities of aldolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase and pyruvate kinase were determined in stimulated rat thymocytes at 24 h intervals during a period of 72 h of culture. In parallel the mRNA levels of these enzymes were analysed by Northern blotting with specific probes. Both the enzyme activities and the corresponding mRNA levels reached their maxima 48 h after stimulation coinciding with the S-phase of the cell cycle. The isozyme types of aldolase and hexokinase in resting and in mitogen-stimulated rat thymocytes were identified by Northern blot hybridisation using isozyme-specific probes. In these cells the aldolase A is expressed, whereas type B and C could not be detected. The transcription of the aldolase A gene can be regulated by two different promoters. Depending on the alternative usage of the promoters the aldolase A-specific mRNA either contains the non-translated exons M1 or AH1. In rat thymocytes the promoter proximal to the exon AH1 is used while the expression of mRNA I, the type characteristic for muscle tissue, was not observed. In contrast to aldolase two isozyme types of hexokinase were detected. Hexokinase I as well as hexokinase II were present in thymocytes whereas hexokinase III was not detectable. A shift in the isozyme pattern was not observed during the cell cycle progression.
Subject(s)
Cell Cycle , Fructose-Bisphosphate Aldolase/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Hexokinase/metabolism , Isoenzymes/metabolism , Thymus Gland/enzymology , Animals , Base Sequence , Cell Division , DNA Primers , Fructose-Bisphosphate Aldolase/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glycolysis , Hexokinase/genetics , Isoenzymes/genetics , Kinetics , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Thymus Gland/cytologyABSTRACT
The activity profile of poly(ADP-ribosyl)transferase was assayed during a complete cell cycle of rat thymocytes stimulated in the presence of interleukin-2 by concanavalin A or monoclonal antibodies against the T-cell antigen receptor (TCRmAB). Poly ADP-ribosylation was measured in permeabilized cells by the incorporation of [adenine-3H]NAD+ into protein bound poly ADP-ribose. The polymers of ADP-ribose were separated from the monomers using dihydroxyboronyl-Bio-Rex 70 columns. The rate of poly(ADP-ribosyl)ation increases during the G1 phase with a maximum 12 h after stimulation. This increase in activity is due to enhanced de novo synthesis of poly(ADP-ribosyl)transferase which can be abolished by the addition of cycloheximide. The half-life of this enzyme during the induction period was estimated to be 4 h. A second activity peak appears during the S-phase of the cell cycle 48 h after stimulation. The maxima of the poly(ADP-ribosyl)ation rate coincide with elevated immunoreactive enzyme levels at 12 and 48 h of culture assayed by Western blotting. The mRNA levels of pADPRT do not correlate with the first maximum of activity, whereas the second maximum was accompanied by a 5-fold increase of the specific mRNA. These results suggest a translational regulation of pADPRT in the G1 phase of the cell cycle, whereas the second activity peak in the S-phase is due to an increased transcription and translation. The induction of pADPRT activity in the G1 phase of TCRmAB-stimulated cells points to a function of poly(ADP-ribosyl)ation in the proliferation of thymocytes.
